The Armadillo as a Model for Peripheral Neuropathy in Leprosy

Richard W. Truman, Gigi J. Ebenezer, Maria T. Pena, Rahul Sharma, Gayathriy Balamayooran,Thomas H. Gillingwater, David M. Scollard, Justin C. McArthur, and Anura Rambukkana

Abstract

Leprosy (also known as Hansen’s Disease) is a chronic infec-tious disease caused byMycobacterium leprae that primarilytargets the peripheral nervous system; skin, muscle, and oth-er tissues are also affected. Other than humans, nine-bandedarmadillos (Dasypus novemcinctus) are the only naturalhosts of M. leprae, and they are the only laboratory animalsthat develop extensive neurological involvement with thisbacterium. Infection in the armadillo closely recapitulatesmany of the structural, physiological, and functional aspectsof leprosy seen in humans. Armadillos can be useful modelsof leprosy for basic scientific investigations into the patho-genesis of leprosy neuropathy and its associated myopathies,as well as for translational research studies in piloting new

diagnostic methods or therapeutic interventions. Practicaland ethical constraints often limit investigation into humanneuropathies, but armadillos are an abundant source ofleprotic neurologic fibers. Studies with these animals mayprovide new insights into the mechanisms involved in lepro-sy that also might benefit the understanding of other demye-linating neuropathies. Although there is only a limitedsupply of armadillo-specific reagents, the armadillo wholegenomic sequence has been completed, and gene expressionstudies can be employed. Clinical procedures, such as elec-trophysiological nerve conduction testing, provide a func-tional assessment of armadillo nerves. Avariety of standardhistopathological and immunopathological procedures in-cluding Epidermal Nerve Fiber Density (ENFD) analysis,Schwann Cell Density, and analysis for other conserved cel-lular markers can be used effectively with armadillos andwill be briefly reviewed in this text.

Key Words: armadillo; ENFD; gene-expression; leprosy;myopathy; neuropathy; Schwann cell; translational

Introduction

L eprosy is a chronic infectious disease caused byMycobacterium leprae. It primarily affects the peripher-al nervous system (PNS) and involves skin, skeletal,

muscle, and other tissues. The distinct nerve involvementduring M. leprae infection is directly associated with the re-markable capacity of M. leprae to invade adult Schwanncells, the glial cells of the PNS, which enclose and supportthe axons of sensory and motor neurons. Schwann cell infec-tion causes complex biological and pathological alterationsincluding demyelination, de-differentiation, and reprogram-ming of the Schwann cells. Infection eventually brings aninflammatory response, which causes nerve injury. Theseevents, orchestrated by M. leprae and the immune responseto it, underlie the extreme disability and gross deformitysometimes associated with this disease, and are the centralfeature in the pathology of leprosy (Scollard et al. 2006).Despite the numerous studies from patients, and cellular bi-ology studies of M. leprae infection in vitro, relatively littleis known about the pathogenesis of leprosy. Large gaps inthe understanding of neuropathogenesis, in particular the

Richard Truman, PhD, is Chief of the Microbiology Section in the Laborato-ry Research Branch of the Department of Health and Humans Services,Health Resources and Services Administration, Healthcare Systems Bureau,National Hansen’s Disease Program, Baton Rouge, Louisiana (HHS\HRSA\HSB\NHDP). Gigi J. Ebenezer, MBBS, MD, is an Assistant Professor inthe Department of Neurology at The Johns Hopkins School of Medicine inBaltimore, Maryland. Maria Pena, DVM, PhD, is a Senior Post DoctoralFellow for the HHS\HRSA\HSB\NHDP and Department of PathobiologicalSciences at the Louisiana State University School of Veterinary Medicine,in Baton Rouge, Louisiana. Rahul Sharma, PhD, is a Senior Post DoctoralFellow for the HHS\HRSA\HSB\NHDP and Department of PathobiologicalSciences at the Louisiana State University School of Veterinary Medicine,in Baton Rouge, Louisiana. Gayathriy Balamayooran, DVM, PhD, is a Post-Doctoral Fellow for the HHS\HRSA\HSB\NHDP and Department of Patho-biological Sciences at the Louisiana State University School of VeterinaryMedicine, in Baton Rouge, Louisiana. Thomas H. Gillingwater BSc, MBA,PhD, is a Professor of Neuroanatomy at the School of Biomedical Sciences,University of Edinburgh, Edinburgh, UK. David Scollard, MD, PhD, is theChief of the Clinical Branch of the Department of Health and Humans Ser-vices, Health Resources and Services Administration, Healthcare SystemsBureau, National Hansen’s Disease Program, Baton Rouge, Louisiana. Jus-tin C. McArthur, MBBS, MPH, FAAN, is a Professor of Neurology, Pathol-ogy, Medicine, and Epidemiology and the Director of the Department ofNeurology at The Johns Hopkins School of Medicine in Baltimore, Mary-land. Anura Rambukkana, PhD, is a Professor and Chair of Regeneration Bi-ology at the MRC Center for Regenerative Medicine, and the Center forNeurodegeneration, and the Center for Infectious Diseases, at the Universityof Edinburgh, Edinburgh, UK.

Address correspondence to Dr. Richard Truman, National Hansen’sDisease Program, Laboratory Research Branch, Louisiana State UniversitySchool of Veterinary Medicine, Skip Bertman Drive, Baton Rouge,Louisiana 70803, or email rtruman@HRSA.gov.

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early preclinical events, have severely impeded progress to-wards developing effective early diagnostics and therapeu-tics for the management of nerve damage in leprosy patients.M. leprae targets the nerves early after the onset of infec-

tion. Peripheral nerves not only serve as the principal targetfor M. leprae infection, but also serve as a safe haven for thebacillus. The blood-nerve barrier protects the organismfrom many host immune responses.M. leprae appears to takeadvantage of the remarkable regenerating capacity of the adultPNS when securing its preferred niche, and regeneration ofdamaged peripheral nerves continues post-treatment in pa-tients with advanced leprosy (Miko et al. 1993). Nerve dam-age progresses gradually over the entire course of the disease.Nerve involvement in leprosy patients can be identified in

the form of sensory and/or motor neuron damage. The dis-ease process is progressive and can lead to deformities anddisabilities unless the patient is treated to reduce the bacterialload, and inflammatory insults are controlled. Although bac-terial cure can be achieved by successful multidrug antimi-crobial therapy, neurological injury continues to occur inpatients and is exacerbated during pathological perturbationsof the host immunological response to M. leprae, known asleprosy reactions. The cause and treatment of these reactionsis quite problematic, and can result in significant injury tothe underlying tissues.There are no laboratory tests to aid early detection of

leprosy, and the disease can only be diagnosed once clinicalsymptoms appear. The incubation period in humans is usuallyestimated to be from 3–5 years, but much longer intervals havebeen described. The clinical presentation is comprised mainlyof skin lesions with characteristic hypoesthesia and anesthesia;illustrating nerve involvement in even the earliest clinicalstages of the disease. The clinical aspects of leprosy and cellbiology of earlyM. leprae infection have been reviewed pre-viously (Rambukkana 2004, 2010; Scollard et al. 2006).Leprosy is curable with combination multi-drug therapy.

Early detection and treatment are the most effective means toavoid its undesirable sequelae. However, the treatment inter-val is protracted and requires months to years to complete.Even after completion of effective antibacterial therapy, ba-cillary clearance from the involved tissues is quite slow. Theresidual dead bacilli retained in tissues provide chronic im-munological stimulation and can be problematic for diseasemanagement (Scollard et al. 2006). The World Health Orga-nization has recorded remarkable reductions in global preva-lence of leprosy over the last few decades, but nearly aquarter of a million new leprosy cases continue to be report-ed globally each year (these estimates are probably low).Many of these individuals present with permanent nervedamage, and there are several million people living todaywith advanced disabilities owing to their leprosy (WorldHealth Organization 2012).Susceptibility to leprosy is rare, and appears to have a ge-

netic component (Alter et al. 2011). Infected individualsmanifest their disease over a broad clinical and histopatho-logical spectrum that is determined by the individual’simmunological response to M. leprae. On one end of the

spectrum (lepromatous) there may be numerous diffuse le-sions, which contain large numbers of bacilli in poorly orga-nized granulomas; showing evidence of little cell-mediatedimmune response to M. leprae. On the other extreme(Tuberculoid) there may be one or few clearly definedlesions with well-organized granulomas showing few, if any,visible bacilli and evidencing active cell mediated immunity.Between these two poles, borderline forms blend the ex-treme forms to make 2–3 additional classifications. However,the common feature across all forms of the leprosy spectrumis focal insensitivity of the lesions, and impaired sensoryand/or motor function resulting from involvement ofM. lep-rae along with damage to the underlying peripheral nerves(Scollard et al. 2006).

M. leprae likely enters the human body through respirato-ry routes and disseminates hematogenously to manifest awidespread, asymmetrical disease pattern. Intraneural infec-tion by M. leprae is the pathognomonic feature of leprosy,and perineural and intraneural inflammation are its morpho-logical hallmarks. The granulomas, which form in thesenerves, are identical in structure to those in skin lesions. Thebacilli proliferate in macrophages and Schwann cells of theperipheral nerves, especially in the hands, legs, and feet, orcooler regions of the body, as M. leprae prefers cooler tem-peratures for growth. Inflammation in the cutaneous nervesof skin lesions may extend proximally for variable distancesand involve other nerve trunks and branches. There may bepronounced edema and fibrosis (Scollard 2008). Somesuperficial nerves can become readily palpable, and high res-olution sonography has been used to quantify and detectnerve damage in some leprosy patients (Jain et al. 2009).The elevation of circulating levels of pro-inflammatory cyto-kines and chemokines have been related to the onset of lep-rosy reactions. Studies of limited biopsy material fromleprosy patients with these reactions indicate that levels ofTNFα in cutaneous nerves are similar to those in the skin(Khanolkar-Young et al. 1995), but little is known aboutgene expression in leprotic nerves. Ethical and practical con-straints generally preclude biopsy of affected human nerves.Most of the clinical understanding about leprosy neuropathyhas been derived from examining amputated tissues takenfrom very advanced stages of infection (Scollard 2008).

Studies with primary nerve tissue culture and mouse mod-els have yielded insight into some molecular interactions ofM. leprae with Schwann cells, which likely facilitate the lo-calization ofM. leprae to the peripheral nerve and benefit itsproliferation in that privileged niche (Rambukkana 2010).Attachment of M. leprae to the basal lamina that surroundsSchwann cell-axon units of nerve fibers seems to be mediat-ed through the G domain of the laminin alpha-2 chain and al-pha dystroglycan receptors on the cell surface (Rambukkanaet al. 1997, 1998; Rambukkana 2000). Interestingly, the ba-cilli also bind and activate the receptor tyrosine kinaseErbB2, which induces the Erk1/2 signal transduction path-ways and results in demyelination and dedifferentiation ofthe terminally differentiated Schwann cell (Noon and Lloyd2005; Rambukkana 2004, 2010; Tapinos and Rambukkana

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2005; Tapinos et al. 2006;). Recent evidence also suggeststhat these dedifferentiated cells are easily parasitized, and thereprogramming of infected Schwann cells to stem cell-likecells provides them a convenient vehicle to promote thespread to other tissues, including skeletal muscles (Masakiet al. 2013). However, translating this knowledge into thedevelopment of new diagnostics or therapeutic applicationsrequires recapitulation of the events in an animal modelsystem, which can validate the findings in the setting of anatural infection.

Management of leprosy patients requires careful monitor-ing of sensory and motor nerve function. Assessment of tac-tile sensory function in leprosy is most routinely measuredusing Semmes-Weinstein monofilaments (MFT), and motorfunction is assessed by standard voluntary muscle tests(VMT). Recent studies examining the development and pro-gression of nerve injury in large numbers of leprosy patientshave shown that impairment begins early and progresseschronically over the course of the disease. Electrophysiologi-cal nerve conduction studies, used in combination withwarm perception threshold testing, and other procedures, canbe used effectively to detect sensory and motor nerve func-tion impairment, as well as predict future clinical abnormali-ties in sensory and motor nerves (Nicholls et al. 2005; Smithet al. 2009; van Brakel et al. 2005, 2008; Wilder-Smith andvan Brakel 2008;).

Investigation of the various components of the nociceptivefibers that are associated with sensory loss in leprosy skin le-sions is scant and sketchy. Tactile sensitivity is mediated bythickly myelinated Aβ fibers of the dermis, while thermalsensitivity is mediated by thin myelinated Aδ fibers and un-myelinated C type fibers that end in the epidermis as freenerve endings (Ebenezer et al. 2007). There is some evi-dence suggesting that the C fibers in the epidermis are theearliest to undergo degeneration, this is consistent with clini-cal observations that the assessment of thermal sensation canbe a primary component in establishing early diagnosis (vanBrakel et al. 2005, 2008).

Peripheral neuropathy in leprosy patients also contributes tothe impairment of skeletal muscle function. Studies on the in-volvement of skeletal muscle in leprosy patients have clearlydemonstrated reduced motor nerve conduction velocities,decreased compound muscle action potentials, and sometimesa complete absence of potentials (Werneck et al. 1999). How-ever, it is unknown howM. leprae infection causes the impair-ment of motor functions, with subsequent muscle atrophy. Inleprosy patients, disseminated M. leprae are detected inskeletal muscles, which are also known to serve asM. lepraereservoirs and play a key role in leprosy pathogenesis (Kauret al. 1981; Werneck et al. 1999). In patient studies, plantarnerves that innervate intrinsic muscles of the foot also showloss of sensation and muscle function impairment, indicatingthat muscle pathogenesis is a key feature in human leprosy.Thus, the involvement of skeletal muscle in human leprosymay occur as a secondary event, resulting from peripheralneuropathy-induced denervation (Pearson et al. 1970), or maybe due to primary lesions in the muscle itself.

M. leprae is an obligate intracellular parasite that cannotbe cultivated on artificial laboratory media. Its long doublingtime (12.5 days) severely limits the utility of many cell cul-ture techniques. The organism prefers cool temperatures, andviability of M. leprae decreases quickly at temperaturesabove 35°C (Truman and Krahenbuhl 2001). Ever since dis-covery of the organism, investigators have attempted to prop-agate M. leprae in a variety of different animal species,especially those with body temperatures lower than 37°C.However, the susceptible animal host range appears to bequite narrow. Most animals readily clear the bacilli (Couret1911; Fite et al. 1964). Limited replication can be achievedafter inoculation ofM. leprae into the hind foot pads of con-ventional mice (Shepard 1960). Although the infectionremains localized to the foot, the level of growth achieved infoot pads is markedly enhanced among athymic nude miceand other immune-deficient mouse strains (Colston andHilson 1976). However, the only immunologically intactanimal which reliably becomes infected withM. leprae, andclosely recapitulates leprosy, is the nine-banded armadillo(Dasypus novemcinctus).

The Armadillo

Armadillos are exotic looking animals about the size of thecommon housecat (Figure 1A). They have short legs, withstrong claws and a hard flexible carapace that armors most oftheir body. They are mammals of the Order Xenarthra; rela-tives of sloths and anteaters. The armadillo’s normal bodytemperature ranges from 33–35°C, and it was this trait thatfirst attracted the attention of leprosy researchers. Experi-mental infection of armadillos with M. leprae requires18–24 months to manifest as a fully disseminated disease,but prolific quantities of bacilli accumulate throughout theanimal’s reticuloendothelial organs and up to 1012 M. lepraecan be harvested from the tissues of a single animal. Theremarkable quantities of M. leprae, made available througharmadillos, have been a boon to leprosy research, and arma-dillos are the hosts-of-choice for in vivo propagation of bulkquantities of leprosy bacilli (Truman and Sanchez 1993).Shortly after initial discovery of the armadillo’s unique

susceptibility to experimental infection, a naturally occur-ring, systemic mycobacteriosis was found among free rang-ing armadillos (Walsh et al. 1975). Subsequent surveysconfirmed that wild armadillos are reservoir-hosts and hadharbored M. leprae for many decades prior to their ever be-ing used in leprosy research (Truman 2005; Walsh et al.1986). Leprosy was not present in the New World duringpre-Colombian times, making it reasonable to assume thatarmadillos have acquired the infection from humans some-time in the last few centuries. They are recognized as theonly nonhuman reservoir of M. leprae, and are part of thenatural ecology of the disease in the United States. Recent re-ports indicate that zoonotic transmission of M. leprae fromarmadillos is responsible for up to 64% of all leprosy casesseen in the United States. These animals might play a

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role perpetuating leprosy elsewhere in the Americas. Besideshumans, armadillos are the only known natural hosts ofM. leprae (Truman et al. 2011).M. leprae infection in the armadillo closely recapitulates

many of the structural, physiological, and functional aspectsof leprosy seen in humans. Armadillos do not exhibit manycharacteristic gross skin lesions. A hard, flexible carapaceobscures the majority of the armadillo’s body; greater in-volvement of the disease is seen internally. However, thefeet, nose, and eyes may show asymmetrically distributed,nonspecific, focal, ulcerative dermatitis that is associatedwith regional insensitivity of the skin. Although the majorityof armadillos appear to be susceptible to experimentalinfection, 15–20% readily resist the challenge. Their granu-lomatous response toM. leprae also appears to be histopath-ologically identical to that of humans; and armadillos exhibita similar spectrum of histopathological responses to theorganism. Although 70% of the animals manifest aLepromatous-type of response to M. leprae, some animalsproduce full Tuberculoid or Borderline forms of the disease(Job et al. 1983; Job and Truman 2000). Beyond sharing aunique susceptibility to M. leprae, perhaps the most im-portant similarity between humans and armadillos is thatthey both develop extensive neurological involvement withM. leprae. No other laboratory animal (e.g., mice, rabbits, and

guinea pigs) develops neurological involvement withM. leprae.The infection path in the armadillo provides a unique opportuni-ty to model the neuropathogenesis of leprosy.

Effective animal models can help provide pivotal new un-derstandings about the mechanisms involved in the diseaseprocess, and the animals may serve as useful platforms forpiloting new therapeutic interventions. Armadillos are wellestablished as models for leprosy pathogenesis and they arethe most abundant source of leprotic neurological fibers forbasic science investigations. Among the armadillo’s uniqueattributes are a controlled and known infection status, com-pressed disease duration, and a functional recapitulation ofleprosy as is seen in humans. Armadillos specimens can beexamined and compared in regards to rare neurologicalevents that happen in both normal and leprotic tissues, allfrom time periods and in material quantities that cannot beobtained from human subjects. Studies with armadillos mayhelp identify new targets for interventions, establish end-points that can be useful for monitoring the clinical course ofinfection in patients, and in evaluating the effectiveness ofnew therapies.

Although armadillos have been used in leprosy researchfor nearly 40 years, their exotic nature and lack ofarmadillo-adaptable research reagents has limited their useas model hosts. Recent completion of the armadillo whole

Figure 1: Collage of armadillo, nerves and Schwann cell involvement. (A) Photo, nine-banded armadillo (Dasypus novemcinctus). (B) Elec-tron micrograph illustratingM. leprae within armadillo Schwann cell. Bar is 500nm. (C) Fite-stained armadillo post-tibial nerve 60x magnifi-cation showing involvement ofM. leprae in vascular endothelial cells, with inset at 120x magnification. (D) Fite-stained armadillo post-tibialnerve 60x magnification showing extensive perineural and endoneural involvement of M. leprae with epineural inflammation. Inset at 120xmagnification illustrates endoneural involvement.

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genomic sequence is now facilitating the development ofarmadillo-specific reagents and molecular probes that areneeded for modern neuropathogenesis studies. This willpropel armadillos as effective models for translational re-search in leprosy. The development of armadillos for usein in vivo propagation of M. leprae and representativemodel hosts of leprosy has been reviewed before (Adamset al. 2012; Scollard and Truman 1999; Sharma et al.2013). This paper focuses on the special advantages of thearmadillo model with regards to enhancing the under-standing about the pathogenesis of leprosy, highlightingsome novel insights into leprosy that have been gainedthrough work with armadillos, and emphasizing end-points and select methods that can be useful for conduct-ing intervention studies with armadillos.

Preclinical Leprosy

A notable advantage when using the armadillo as a model isthe opportunity to examine the pathogenesis of infection atpreclinical stages that have never been observed in humans,which are more likely to be effectively targeted by therap-eutic intervention. Following experimental infection ofarmadillos,M. leprae populates the peripheral nerves and re-ticuloendothelial tissues; then slowly disseminates systemi-cally from these early foci. The armadillo post-tibial nerveruns for about 5 cm beneath the skin surface of the medialhind limb. This nerve has a high frequency of involvementin both armadillo and human infections. It is easily accessi-ble in the armadillo and a useful target for studies in armadil-los. Although the duration of experimental infection inarmadillos (4–24 months) is highly compressed when com-pared to the many years involved in human infections, bacil-lary loads of ≥106 M. leprae/cm are common in armadillopost-tibial nerves (Figure 1).

The first essential step in leprosy neuritis is the localiza-tion ofM. leprae to the peripheral nerve. Detailed histopath-ological studies on armadillo post-tibial nerves suggests thatthis localization is a multistep process, which proceeds fromthe outside-in as opposed to an ascending-type of infectionthat is sometimes described in the early literature. The bacillifirst aggregate in epineural lymphatics and blood vessels,then enter the endoneural compartment through its bloodsupply (Scollard et al. 1999; Scollard 2000). This view givessignificance to old observations of substantial involvementof endothelial cells, and suggests that the substantial perineu-ral inflammation seen in leprosy is evidence when trackingthe route of infection in the nerves themselves (Figure 1B,C, and D). In addition to implications with respect to the un-derstanding of neuropathogenesis, these studies highlightseveral additional possible points of preventative, or thera-peutic, intervention that might otherwise remain ignored.These points include: interrupting M. leprae binding to en-dothelial cells, entry into endothelium, exit from endothelialcells through the perineurium and into the endoneurium, andbinding to Schwann cells. Any of these factors could be

critical points to potentially negate establishment of infectionin the nerve and eventual nerve injury (Scollard 2008).

Molecular Studies on Clinically RelevantNerves

Once M. leprae populates the nerve the bacilli can growto high numbers and spread to adjacent nerve trunks. Anti-leprosy drug therapies must have good neural penetration inorder to kill bacilli sequestered in nerves. Even once effec-tively killed by the anti-microbial drugs, humans and arma-dillos show only slow clearance of bacilli from nerves andskin lesions. In one study, ten M. leprae infected armadilloswere allowed to incubate their infections for twelve months,before five of them were treated with 10mg/kg rifampin oncemonthly per os for an additional twelve months. These andan additional five naïve control animals were later sacrificedat twenty-four months postinfection. Although each of thetreated animals showed clinical improvement in skin lesionsand ulcers as a result of the antimicrobial therapy, examina-tion of their post-tibial nerves showed continued presence ofM. leprae.Molecular assessment ofM. leprae viability sug-gested the organisms had been effectively killed by rifampintherapy. However, bacterial counts averaging 104–5 bacilliper cm of post-tibial nerve were still observed even after theconclusion of a full year of treatment (Figure 2). This heavyburden of (dead) bacilli provides a rich substrate for contin-ued immunological interaction with the host, and suggeststhere is insidious chronic injury to nerves involved withM. leprae.Although there are no comparable human studies, arma-

dillo nerve segments can be used effectively for gene expres-sion profiling and analysis of cell signaling pathways. Thegene expression profiles between uninfected-normal nerves,and infected-untreated or infected-rifampin-treated armadillopost-tibial nerves can be compared with a broad array ofneural specific markers. In examining the nerves describedabove, the gene expression profiles reflected ongoing degen-eration and regeneration processes among the infectedanimals when compared to the naïve controls, as well as evi-dence of persistent inflammation with enhanced expressionof both IFNg and TNFa (Figure 3). Though gene expressionamong treated animals was somewhat lower than those ofthe animals suffering active infection, the gene expressionprofiles of nerve segments from rifampin treated animalsmore closely resembled those of the untreated animals overthe naïve controls (Figure 3). The slow clearance of killedbacilli can be problematic for nerve injury. Molecular mark-ers for neurodegeneration and regeneration, along with geneexpression profile of inflammatory genes, and enumerationof the bacterial load ofM. leprae in the nerve are useful ther-apeutic end-points for laboratory studies. They highlightthe importance of developing new therapies to enhanceclearance of bacilli from the host in conjunction with anti-bacterial treatment in order to limit the progress of insidiousneuropathy.

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Electrophysiological Studies

Armadillos do not reliably respond to thermal, light, or tac-tile nociceptive stimulants, but measurements of nerve con-duction can be used effectively to assess function ofarmadillo motor nerves. Nerve conduction studies are nonin-vasive, and are ideal for repeated, or prospective, assess-ments studying the onset and progress of peripheralneuropathy over time in the same subject. Normal armadillosexhibit conduction profiles similar to humans (mean NCV62.09 ± 10.72 m/sec, mean CMAP 1.55 ± 0.33 mV). De-myelinating events result in a decreased Nerve ConductionVelocity (NCV) measured in m/sec, while axonal lossand muscular atrophy leads to a decrease in the CompoundMotor Action Potential (CMAP) measured in mV (Franssen2008). Conduction deficit is observed in the post-tibialnerves of 75% of all experimentally infected armadillos,with onset occurring as early as 90 days postinfection.

Similar to observations on humans, depressed CMAPamplitude (<0.9 mV, mean – 2 SD) is the most common pre-sentation, but abnormal nerve conduction velocity (NCV<40m/s, mean -2SD) also can be observed. Most armadillosprogress from normal conduction to a total conduction blockby the latest stages of their experimentally induced infectionswith M. leprae. Onset of conduction abnormality generallycoincides with evolution of a detectable immune response toM. leprae (i.e., detectable PGL1 IgM antibodies) and is asignificant predictor of other non-specific symptoms of neu-ropathy such as foot ulcers, and nail avulsion or hypertrophy(Sharma et al. 2013). Unfortunately, their hard carapace andthick skin limit the number of nerves that may be examinedin armadillos, and sensory nerve conduction profiles havenot yet been successful. However, other histopathologicaltechniques can be substituted effectively.

Epidermal Nerve Fiber and SchwannCell Density

The morphological and quantitative study of skin biopsyoffers an alternative tool to assess thin nerve fiber structurerelated to the thermal sensitivity function. Immunostaining ofpunch skin biopsies for protein gene product 9.5 (PGP9.5), aneuronal pan axonal marker, has now been used by severalinvestigators to visualize the intra-epidermal nerve fibers, der-mal nerves, and Schwann cells in lieu of nerve conductiontests which may fail to detect small nerve fiber impairment.The small fiber innervation is length dependent and robustnormative data for epidermal nerve fiber densities (ENFD) inthe distal limb have been developed (McArthur et al. 1998).In small fiber sensory neuropathies associated with diabetes,HIV, and idiopathic small fiber sensory neuropathies, a de-crease in epidermal density in the distal leg has been demon-strated (Goransson et al. 2006; Holland et al. 1997; Periquetet al. 1999; Polydefkis et al. 2004). Abnormalities were dem-onstrated in cutaneous innervation even in individuals withnormal tendon reflexes at the ankles, normal sural nerve ac-tion potential amplitudes, and normal quantitative sensorytests (Gibbons et al. 2006). Although leprosy neuritis hasbeen well described clinically and histologically, the underly-ing mechanisms of nerve damage remain poorly understood.Very little morphological work has been done to detect earlydamage to sensory nerves.

Quantitation of epidermal fibers in skin biopsies of ears,abdomen, and a distal leg of naïve armadillos has shown alength dependent innervation similar to humans (GJ Ebe-nezer, R Truman, D Scollard, M Polydefkis, R Lahiri, JCMcArthur, unpublished data). The epidermal innervationwas extremely dense in the ear and abdomen compared tothe distal leg (Figure 4A, B, and C) and the lower limit ofnormal (defined as the 5th percentile) was 21.3 fibers/mm atthe distal leg. The ENFD in armadillos was higher in com-parison to the normative densities published in humanhealthy controls (Lauria et al. 2010). The infected animals

Figure 2: Comparison of M. leprae infiltrated to armadillo post-tibial nerves among treated and untreated animals. Mean and Stan-dard deviation ofM. leprae/cm of armadillo post-tibial nerves (fiveanimals each group) harvested 24 months postinfection and seg-mented to an equal distal and proximal portion. Treated animals re-ceived 12 months anti-microbial therapy, with once monthly10 mg/kg rifampin per os. Nerve trunks from Treated and Untreatedanimals were segmented to distal and proximal portions and pro-cessed for simultaneous DNA and RNA extraction. Bacteria wereenumerated and normalized per segment centimeter, and valuespooled to derive mean and standard deviation. Untreated with anadditional 12 months of incubation showed higher overall numbers,but treated animals also retained large numbers of deadM. leprae intheir nerves, even one year after therapy was initiated.Total numberof leprosy bacilli in each segment were enumerated usingM. leprae,specific quantitative PCR assay targeting RLEP gene and numberof viable only bacilli were calculated by using quantitative RT-PCRassay targeting 16S rRNA (Truman et al. 2008). Although, no via-ble bacilli were detected after one year Rifampin treatment, up to10E + 04 dead bacilli were still not cleared from the treated armadil-lo nerves. Bacterial counts were significantly (p < 0.05) higher insegments of distal half, compared to proximal half of PT nerve har-vested from infected (p = 0.0023) animals as well as after treatment(p = 0.019).

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showed a lower mean ENFD compared to naïve animals,suggesting early small fiber degeneration. Double stainingof cutaneous axons and Schwann cells in naïve armadillosalso mimicked the human cutaneous nerve network pattern

(Figure 4D). Schwann cells of dermal cutaneous nerves ininfected armadillos showed a trend towards increasing den-sity (GJ Ebenezer, R Truman, D Scollard, M Polydefkis,R Lahiri, JC McArthur, unpublished data) and thus provided

Figure 3: Gene expression profiling of Naïve,M. leprae-Infected Untreated and Treated armadillo post-tibial nerves. Expression of molecularmarkers associated with nerve structure and function (PGP9.5 [UCHL1] PMP 22, β- tubulin, Neurofilament, NCAM, growth factors [NGF βand DLK-1,]) and inflammation (TNF-α and IFN-γ) was compared between armadillo post-tibial nerves harvested from 1) Naïve: uninfected-normal animals, 2) Infected Untreated animals, and 3) Treated, Infected animals that had received 12 months of rifampin treatment. Data was nor-malized using GAP3DH, and relative expression was computed by using the ΔΔCt method. Results represent mean ± SD from duplicate experi-ments on five animals in each group.

Figure 4: Skin sections (A, B, and C) immunostained with anti-PGP 9.5, neuronal marker, and double stained confocal image (D) of axons(anti-PGP 9.5) and Schwann cells (anti nerve growth factor receptor, p75). (A), (B), and (C): Skin sections from the ear lobes (A) and abdomen(B) of naïve animals showing dense epidermal innervation (black arrows), compared to the distal leg (C). (D): Distal leg section from a naïveanimal exhibiting axons entering the epidermis (black arrow) and the Schwann cells (gray arrow) are en-sheathing and co-localizing (white ar-row) on the dermal axons. Scale bars: (A, B, and C) = 50um and (D) = 20um.

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indirect evidence that during early infection Schwann cellsundergo proliferation while harboring M. leprae. Thoughthis difference was not significant statistically, it reaffirmsthe feasibility of studying small fibers in the armadillo usingthis technique, and the possibility of using it as a novel toolto test new drugs and in therapeutic interventions.

Impairment of Muscle Architecture andFunction in Infected Armadillos

A common pathological hallmark of human leprosy, andM. leprae-infected armadillos, is the involvement of extremi-ties. The lumbrical muscles of the foot are innervated by themedial and lateral plantar nerves, and in leprosy patientsthese plantar nerves are affected. Given that leprosy is a

classic example of an infectious disease that can cause mus-cle paralysis due to neurological injury, the pathological sta-tus of lumbrical muscles from the armadillo hind limb hasbeen examined.

The organization of intact muscle architecture can be eval-uated by labeling muscle tissues with antibodies to adult my-osin, a family of ATP-dependent, actin-binding, and highlyconserved muscle motor protein. Antibodies specific for my-osin heavy chains (myosin HC) that react with mature myofi-brils were used to detect the myosin distribution andarchitecture of lumbrical muscles. Labeling of transversesections of control armadillo muscles showed a highly orga-nized architecture of muscle fibers with clear endomysiumand perimysium (Figures 5A, B, and E), similar to humanand rodent skeletal muscle. In contrast lumbrical musclesfrom infected animals displayed a markedly disorganized

Figure 5: Pathological features of skeletal muscles in infected armadillos. (A-D) Double immunolabeling of transverse sections of control (A,B) and infected (C, D) deep lumbrical muscles showing an accumulation of fibrotic tissues (C, D) and immunolabeling with antibodies to my-osin heavy chain (Myosin HC; green) and laminin or collagen (red), counter stained with DAPI for nuclei (blue). Note the massive number ofnucleated cells present in fibrotic tissues associated with infected muscles that were not present in the control muscles (weak myosin HC stain-ing in green in fibrotic tissues represent non-specific background fluorescence). Also note the disorganized muscle architecture and reducednumber of muscle fibers with accumulated matrix in infected muscles (C and D) as compared to controls (A and B). Higher magnification ofmyosin HC in the boxed areas in (B) and (D) are shown in (E), (control: myosin HC in green), (F) (infected: myosin HC in green) and (G) (in-fected: myosin HC in green and M. leprae labeling by PGL-1 antibody in red counter stained with DAPI for blue nuclei). Note the disorga-nized myosin HC labeling showing disrupted muscle fibers with infiltrated cells (blue nuclei) containing large number of M. leprae (redlabeling in G) infected animals.

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pattern of muscle fibers, with disorganized endomysium andperimysium (Figures 5C, D, and F). Analysis of transversesections of lumbrical muscles with antibodies specific forbasal lamina components, laminin, and collagen showedmarkedly disrupted and abnormal extracellular matrixexpression in infected muscles (Figures 5C and D) as com-pared to control animals, whose labeling was confined to theouter surface of each muscle fiber (Figures 5A and B). Co-localization of laminin and collagen with myosin HC clearlyshowed abnormal accumulation of matrix componentswithin the endomysium, and additional fibrotic growth(Figures 5C and D). Detailed analysis of muscle fibersshowed weak or inconsistent myosin HC expression, with aless striated pattern compared to controls (Figures 5E and F).Nuclear labeling revealed an increased accumulation of cellsin the muscle, most likely mononuclear inflammatory cellsor macrophages. In uninfected animals, nuclear labeling wasfound only adjacent to individual muscle fibers, which issimilar to normal adult mouse and human muscles. Further-more, the distribution ofM. lepraewithin the lumbrical mus-cles in infected animals was studied using antibody (Ab) toM. leprae PGL-1 that specifically detects whole M. leprae(Masaki et al. 2013). These data revealed thatM. lepraewerepredominantly localized to cells in the interstitial tissues inthe perimysium, most likely within the infiltrated cells.

In agreement with these findings, functional studies alsoshowed that the small lumbrical and flexor muscles of the ar-madillo foot have early involvement with M. leprae. Brand(Brand et al. 1981) showed that the PCSA (cross sectionalarea/mass) of muscles in the hands of leprosy patients couldbe used as a surrogate measure of grip strength and indexmuscle atrophy. Examining the PCSA of armadillo small(intrinsic) lumbrical and flexor muscles shows a qualitativereduction of muscle mass among infected armadillos, withPGL1 IgM positive animals having an average of 20% lessmuscle mass than naïve normal animals. Detailed histopath-ological studies showed that long-term infection in the arma-dillo also has discernible effect on the morphological andmolecular composition of skeletal muscle fibers. These fea-tures of the skeletal muscles in infected armadillos resemblesthe muscle pathology and function impairment documentedin patients with lepromatous leprosy with a high bacterialload (Werneck et al. 1999), suggesting the potential ofusing the armadillo model not only for neuropathies but alsomyopathies associated with human leprosy.

Routes and Timing of Infection

Armadillos are susceptible to experimental infection withM. leprae by a variety of routes, including intravenous, intra-dermal, percutaneous, and respiratory instillation (Sharmaet al. 2013; Truman and Sanchez 1993). In addition, manyarmadillo nerve trunks are large enough to permit directinoculation of bacilli to the peripheral nerve. Regardless ofthe route of administration, the type of disease that is mani-fest in the animals depends on that particular animal’s innate

or pre-existing response to M. leprae. Lepromatous-typearmadillos will eventually develop a fully disseminated in-fection, while the level and type of involvement will be lesswith animals that manifest other forms of leprosy.The duration of infection in the armadillo is somewhat idio-

syncratic, but is mainly a factor of the viable challenge dose ofbacilli given. The standard challenge dose is 1 × 109 highly vi-ableM. leprae given intravenously. At this dose most animalswill develop a fully disseminated infection, requiring humaneeuthanasia within 18-24 months. Lower challenge doses re-quire accordingly longer periods to manifest full dissemina-tion (Truman 2008). Studies addressing events of preclinicalleprosy do not require fully disseminated infections, and canbe initiated immediately following challenge.

Limitations of the Armadillo Model

Armadillos are exotic animals and are not commonly used inlaboratory studies outside of leprosy research. The primarylimitation in use of armadillos is the paucity of reagents, es-pecially antibodies, to facilitate investigations. With recentcompletion of the armadillo whole genomic sequence, inves-tigative reagents can be generated more easily. Highly specif-ic molecular probes and primers are readily designed. All ofthese reagents require independent development and verifi-cation of their quality.Armadillos are not available from standard commercial

vendors and must be obtained from the wild for investigativepurposes. Some IACUC or animal facilities may not beequipped to deal with wild animals. In addition, such wildanimals are highly out-bred and may exhibit wide variationsin response to challenge. Armadillos do not breed reliably incaptivity, but female armadillos routinely give birth to mono-zygotic quadruplets, and gravid females captured from thewild will litter in captivity, making it possible to conductstudies on matched sets of identical twins (Truman 2008).

Conclusions

Leprosy is a complex infectious disease that can cause dis-abling damage to peripheral nerves. Although the infectioncan be cured bacteriologically, there are no effective therapiesfor reversing nerve damage or preventing additional complica-tions after therapy. Ethical and practical constraints in studyingthe neurological aspects of human leprosy have left large gapsin the common knowledge of the mechanisms involved in in-ducing neuropathy and few strategies for the development ofnew diagnostic approaches or therapeutic regimen. Studieswith animal models could markedly benefit the human under-standing of the mechanisms of nerve injury in leprosy, andmight provide a useful vehicle for evaluating new therapies.Other than humans, the nine-banded armadillo is the only

animal that develops extensive neurological involvementwith M. leprae. Comparative pathological studies show thatarmadillos closely recapitulate many of the functional, physio-logical, and structural aspects of human leprosy; and a variety

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of routine clinical, diagnostic, and immuno-pathological tech-niques can be employed effectively to monitor and stage M.leprae infections in these animals. In addition, armadillosyield an abundant supply of rare leprotic tissues that canfacilitate molecular and histopathological studies that wouldnot be possible to consider with other human or animalresources.Developing techniques to effectively detect and monitor

the onset and progress of leprosy neuropathy could havesignificant benefit to leprosy patients and may provide addi-tional insights for developing new intervention strategies.The compressed duration of infection in armadillos andgeneral characteristics of the infection in these animalsmake them good candidates for evaluating adjunctiveimmunological and drug therapy combinations, new anti-neuropathic medications, and safety analysis of new drugs orvaccines before they are deployed in human populations.In addition, experimental leprosy can be viewed as an induc-ible neuropathy that results in demyelination of Schwanncells and chronic inflammation of peripheral nerves. Theunderstandings gained from studies with armadillos alsomay be of benefit to research efforts on other demyelinatingneuropathies, and will better integrate leprosy into the largercommunity of important neuropathic diseases.

Acknowledgments

The authors are grateful for the technical assistance of KyleAndrews, Angelina Deming, Vilma Marks, Greg McCor-mick, Roena Stevenson, and Heidi Zhang at the NHDP. Wealso express our appreciation to Karen Burr and Sang SooSeo at the University of Edinburgh for studies on musclepreparation and microscopy. We thank Peter Hauer at theJohns Hopkins School of Medicine for his assistance in thisanalysis. These studies were supported in part by funds fromthe Miss A. M. Urquhart Trust, UK and the University ofEdinburgh, Scotland, UK. As well as the United States De-partment of Health and Human Services, Health Resourcesand Services Administration, Healthcare Systems Bureau,National Hansen’s Disease Program and the National Insti-tutes of Allergy and Infectious Disease (IAA-2646).

References

Adams LB, Pena MT, Sharma R, Hagge DA, Schurr E, Truman RW. 2012.Insights from animal models on the immunogenetics of leprosy: A re-view. Mem Inst Oswaldo Cruz 107 Suppl 1:197–208.

Alter A, Grant A, Abel L, Alcaïs A, Schurr E. 2011. Leprosy as a geneticdisease. Mamm Genome 22:19–31.

Brand PW, Beach BB, Thompson DE. 1981. Relative tension and potentialexcursion of muscles in the forearm and hand. J Hand Surg Am3:209–219.

Colston MJ, Hilson GR. 1976. Growth of Mycobacterium leprae andM. marinum in congenitally athymic (nude) mice. Nature 5567:399–401.

Couret M. 1911. The behavior of bacillus leprae in cold-blooded animals.J Exp Med 5:576–589.

Ebenezer GJ, Hauer P, Gibbons C, McArthur JC, Polydefkis M. 2007.Assessment of epidermal nerve fibers: A new diagnostic and predictive

tool for peripheral neuropathies. J Neuropathol Exp Neurol (66)12:1059–1073.

Fite GL, Wrinkle CK, Sanchez R. 1964. Inoculations of M. Leprae in Rep-tiles. Int J Lepr 32:272–278.

Franssen H. 2008. Electrophysiology in demyelinating polyneuropathies.Expert Rev Neurother 8(3):417–431.

Gibbons CH, Griffin JW, Polydefkis M, Bonyhay I, Brown A, Hauer PE,McArthur JC. 2006. The utility of skin biopsy for prediction of progres-sion in suspected small fiber neuropathy. Neurology 66(2):256–258.

Goransson LG, Brun GJ, Harboe E, Mellgren SI, Omdal R. 2006. Intraepi-dermal nerve fiber densities in chronic inflammatory autoimmunediseases. Arch Neurol 63(10):1410–1413.

Holland NR, Stocks A, Hauer P, Cornblath DR, Griffin JW, McArthur JC.1997. Intraepidermal nerve fiber density in patients with painful sensoryneuropathy. Neurology 48(3):708–711.

Jain S, Visser LH, Praveen TL, Rao PN, Surekha T, Ellanti R, Abhishek TL,Nath I. 2009. High-resolution sonography: A new technique to detectnerve damage in leprosy. PLoS Negl Trop Dis 3(8):e498.

Job CK, Kirchheimer WF, Sanchez RM. 1983. Variable lepromin responseto Mycobacterium leprae in resistant armadillos. Int J Lepr OtherMycobact Dis 51(3):347–353.

Job CK, Truman RW. 2000. Comparative study of Mitsuda reaction to nudemouse and armadillo lepromin preparations using nine-banded armadil-los. Int J Lepr Other Mycobact Dis 68(1):18–22.

Kaur S, Malik AK, Kumar B. 1981. Pathologic changes in striated musclesin leprosy. Lepr India 53(1):52–56.

Khanolkar-Young S, Rayment N, Brickell PM, Katz DR, Vinayakumar S,Colston MJ, Lockwood DN. 1995. Tumour necrosis factor-alpha(TNF-alpha) synthesis is associated with the skin and peripheral nervepathology of leprosy reversal reactions. Clin Exp Immunol 99(2):196–202.

Lauria G, Bakkers M, Schmitz C, Lombardi R, Penza P, Devigili G,Smith AG, Hsieh ST, Mellgren SI, Umapathi T, Ziegler D, Faber CG,Merkies IS. 2010. Intraepidermal nerve fiber density at the distal leg: Aworldwide normative reference study. J Peripher Nerv Syst 15(3):202–207.

Masaki T, Qu J, Cholewa-Waclaw J, Burr K, Raaum R, Rambukkana A.2013. Reprogramming adult Schwann cells to stem cell-like cells byleprosy bacilli promotes dissemination of infection. Cell 152(1-2):51–67.

McArthur JC, Stocks EA, Hauer P, Cornblath DR, Griffen JW. 1998. Epider-mal nerve fiber density: Normative reference range and diagnosticefficiency. Arch Neurol 55(12):1513–1520.

Miko TL, Le Maitre C, Kinfu Y. 1993. Damage and regeneration of periph-eral nerves in advanced treated leprosy. Lancet 342(8870):521–525.

Nicholls PG, Bakirtzief Z, Van Brakel WH, Das-Pattanaya RK, Raju MS,Norman G, Mutatkar RK. 2005. Risk factors for participation restrictionin leprosy and development of a screening tool to identify individuals atrisk. Lepr Rev 76(4):305–315.

Noon LA, Lloyd AC. 2005. Hijacking the ERK signaling pathway:Mycobacterium leprae shuns MEK to drive the proliferation of infectedSchwann cells. Sci STKE 2005(309):e52.

Pearson JM, Rees RJ, Weddell AG. 1970. Mycobacterium leprae in thestriated muscle of patients with leprosy. Lepr Rev 41(3):155–166.

Periquet MI, Novak V, Collins MP, Nagaraja HN, Erdem S, Nash SM,Freimer ML, Sahenk Z, Kissel JT, Mendell JR. 1999. Painful sensoryneuropathy: Prospective evaluation using skin biopsy. Neurology53(8):1641–1647.

Polydefkis M, Hauer P, Sheth S, Sirdofsky M, Griffin JW, McArthur JC.2004. The time course of epidermal nerve fibre regeneration: Studies innormal controls and in people with diabetes, with and without neuropa-thy. Brain 127(Pt 7):1606–1615.

Rambukkana A. 2000. Molecular basis of the interaction ofMycobacteriumleprae with peripheral nerve: Implications for therapeutic strategies.Lepr Rev 71 Suppl:S168–169.

Rambukkana A. 2004. Mycobacterium leprae-induced demyelination: Amodel for early nerve degeneration. Curr Opin Immunol 16(4):511–518.

Volume 54, Number 3, doi: 10.1093/ilar/ilt050 2013 313

by guest on April 9, 2014

http://ilarjournal.oxfordjournals.org/D

ownloaded from

Rambukkana A. 2010. Usage of signaling in neurodegeneration and regen-eration of peripheral nerves by leprosy bacteria. Prog Neurobiol 91(2):102–107.

Rambukkana A, Salzer JL, Yurchenco PD, Tuomanen EI. 1997. Neural tar-geting of Mycobacterium leprae mediated by the G domain of thelaminin-alpha2 chain. Cell 88(6):811–821.

Rambukkana A, Yamada H, Zanazzi G, Mathus T, Salzer JL,Yurchenco PD, Campbell KP, Fischetti VA. 1998. Role of alpha-dystroglycan as a Schwann cell receptor for Mycobacterium leprae.Science 282(5396):2076–2079.

Scollard DM. 2000. Endothelial cells and the pathogenesis of lepromatousneuritis: Insights from the armadillo model. Microbes Infect 2(15):1835–1843.

Scollard DM. 2008. The biology of nerve injury in leprosy. Lepr Rev 79(3):242–253.

Scollard DM, Adams LB, Gillis TP, Krahenbuhl JL, Truman RW,Williams DL. 2006. The continuing challenges of leprosy. ClinMicrobiol Rev 19(2):338–381.

Scollard DM, McCormick G, Allen JL. 1999. Localization of Mycobacteri-um leprae to endothelial cells of epineurial and perineurial blood vesselsand lymphatics. Am J Pathol 154(5):1611–1620.

Scollard DS, Truman RW. 1999. Armadillos as animal models for leproma-tous neuropathy. In: Deason, ed. Animal Models for BiomedicalResearch. New York: Academic Press. p 330–336.

Sharma R, Lahiri R, Scollard DM, Pena M, Williams DL, Adams LB,Figarola J, Truman RW. 2013. The armadillo: A model for the neuropa-thy of leprosy and potentially other neurodegenerative diseases. DisModel Mech 6(1):19–24.

Shepard CC. 1960. The experimental disease that follows the injection ofhuman leprosy bacilli into footpads of mice. Journal of ExperimentalMedicine 112:445–454.

Smith WC, Nicholls PG, Das L, Barkataki P, Suneetha S, Suneetha L,Jadhav R, Sundar Rao PS, Wilder-Smith EP, Lockwood DN, vanBrakel WH. 2009. Predicting Neuropathy and Reactions in Leprosy atDiagnosis and Before Incident Events-Results from the INFIR CohortStudy. PLoS Negl Trop Dis 3(8):e500.

Tapinos N, Ohnishi M, Rambukkana A. 2006. ErbB2 receptor tyrosine ki-nase signaling mediates early demyelination induced by leprosy bacilli.Nat Med 12(8):961–966.

Tapinos N, Rambukkana A. 2005. Insights into regulation of humanSchwann cell proliferation by Erk1/2 via a MEK-independent and

pp56Lck–dependent pathway from leprosy bacilli. Proc Natl Acad Sci US A 102(26):9188–9193.

Truman RW. 2005. Leprosy in wild armadillos. Leprosy Review 76:198–208.Truman RW. 2008. Leprosy. In: Vizcaino SF, Loughry WJ, eds. The Biology

of the Xenarthra. Gainesville: University Press of Florida. p. 111-119.Truman RW, Andrews PK, Robbins NY, Adams LB, Krahenbuhl JL,

Gillis TP. 2008. Enumeration of Mycobacterium leprae using real-timePCR. PLoS Negl Trop Dis 2(11):e328.

Truman RW, Krahenbuhl JL. 2001. Viable M. leprae as a research reagent.Int J Lepr Other Mycobact Dis 69(1):1–12.

Truman RW, Sanchez RM. 1993. Armadillos: Models for leprosy. LabAnimal 22(1): 8-32.

Truman RW, Singh P, Sharma R, Busso P, Rougemont J, Paniz-Mondolfi A,Kapopoulou A, Brisse S, Scollard DM, Gillis TP, Cole ST. 2011. Proba-ble zoonotic leprosy in the southern United States. N Engl J Med 364(17):1626–1633.

van Brakel WH, Nicholls PG, Das L, Barkataki P, Maddali P, Lockwood DN,Wilder-Smith E. 2005. The INFIR Cohort Study: Assessment of sensoryand motor neuropathy in leprosy at baseline. Lepr Rev 76(4):277–295.

van Brakel WH, Nicholls PG, Das L, Barkataki P, Maddali P,Lockwood DN, Wilder-Smith E. 2005. The INFIR Cohort Study: Inves-tigating prediction, detection and pathogenesis of neuropathy and reac-tions in leprosy. Methods and baseline results of a cohort ofmultibacillary leprosy patients in north India. Lepr Rev 76(1):14–34.

van Brakel WH, Nicholls PG, Wilder-Smith EP, Das L, Barkataki P,Lockwood DN, INFIR Study Group. 2008. Early diagnosis of neuropa-thy in ceprosy–comparing diagnostic tests in a large prospective study(the INFIR Cohort Study). PLoS NTD 2(4):e212.

Walsh GP, Meyers WM, Binford CH. 1986. Naturally acquired leprosy in thenine-banded armadillo: A decade of experience 1975-1985. J Leukoc Biol40(5):645–656.

Walsh GP, Storrs EE, Burchfield HP, Cotrell EH, Vidrine MF, Binford CH.1975. Leprosy-like disease occurring naturally in armadillos.J Reticuloendothelial Soc 18(6):347–351.

Werneck LC, Teive HA, Scola RH. 1999. Muscle involvement in leprosy.Study of the anterior tibial muscle in 40 patients. Arq Neuropsiquiatr 57(3B):723–734.

Wilder-Smith EP, Van Brakel WH. 2008. Nerve damage in leprosy and itsmanagement. Nat Clin Pract Neurol 4(12):656–663.

World Health Organization. 2012. Global leprosy situation. Wkly EpidemiolRec 34:317–328.

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by guest on April 9, 2014

http://ilarjournal.oxfordjournals.org/D

ownloaded from